Protein A is coupled to a resin support in order to create protein A affinity chromatography media commonly used in the manufacturing of recombinant therapeutic monoclonal antibodies. Natural protein A is derived from Staphylococcus aureus and contains five homologous antibody binding regions and a C-terminal region for cell wall attachment. In addition to naturally derived protein A, recombinant material manufactured in Escherichia coli, as well as several engineered versions of the protein, also manufactured recombinantly, have entered the market place. When immobilized on a column, protein A provides a highly efficient and robust purification method for purifying antibodies at various scales. However, protein A ligand from the column can co-elute with the antibody during purification, an effect which is often referred to as protein A leaching. This tendency increases as the chromatography medium ages. Engineered versions of protein A may improve the pH tolerance of the medium, but do not eliminate leaching. It is the current regulatory expectation that leached protein A should be cleared during the purification of antibodies for human use, and manufacturing processes should be validated accordingly. Enzyme-Linked Immunosorbent Assay (ELISA)-based residuals testing is generally employed during process development and validation to assure the efficient removal of residual protein A during process steps following protein A affinity chromatography. In addition, the manufacturer should have a clear understanding and documentation of resin and ligand quality through raw materials qualification and column lifetime studies.

Protein A is derived from Staphylococcus aureus. The structure is composed of a single polypeptide chain containing four IgG binding domains. With the exception of IgG3, all other human IgGs bind to protein A. Each molecule of Protein A is capable of binding two IgG molecules. It is manufactured as a bulk solution at a concentration of greater than 20 mg protein A per mL with an IgG-binding potency of greater than 95%. Because Protein A is used as an ancillary material in the manufacture of recombinant therapeutic drugs, regulatory requirements differ from those for therapeutic drug products.

Packaging and storage
Store in closed containers at the temperature indicated on the label.

Labeling
Preserve in sealed containers, and store at a temperature of 20 or below.

A: SDS-PAGEIt meets the requirements of Identification test A under rProtein A using USP Protein A RS.

B: IgG BindingIt meets the requirements of Identification test B under rProtein A using USP Protein A RS.

Microbial limits 61
The total aerobic microbial count does not exceed 100 cfu per mL, and the total yeasts and molds count does not exceed 10 cfu per mL.

Bacterial endotoxins 85
It contains not more than 1 USP Endotoxin Unit per mg of total protein. [NoteThe Bacterial endotoxins test for Protein A is used to describe the quality of this ancillary material. This test does not define the acceptable level of bacterial endotoxin in the preparation of injectable dosage forms in which Protein A is used.]

in which A is the absorbance of Protein A at the wavelength of 275 nm and 0.149 is the molar absorptivity. Average the triplicate results, and determine a coefficient of variance (CV): the CV is 5%.

Limit of common contaminant protein and corresponding assay

Enterotoxin B
Enterotoxin B is determined using a commercially available microstrip enzyme-immunoassay kit.1 Wells of the microstrips are coated with sheep antibodies to enterotoxin B. Standard curves are made using the ELISA kit control. The negative controls are wells coated with serum from nonimmunized sheep. The level of enterotoxin is determined from the standard curve. The specification for the enterotoxin B level is 1 ng per mg of total protein.

Mobile phase
Prepare a solution of 50 mM sodium dihydrogen phosphate, pH 6.5 in the following manner. Add 6.9 ± 0.1 g of sodium dihydrogen phosphate into a 1000-mL beaker. Dilute with water to 900 mL, and adjust with 5 M sodium hydroxide to a pH of 6.50 ± 0.05. Transfer the solution into a 1000-mL volumetric flask, and dilute with water to volume. Pass the solution through a 0.22-µm membrane filter.

Column regeneration solution
Prepare a solution of 0.1 M sodium dihydrogen phosphate, pH 3.0 in the following manner. Add 13.8 ± 0.1 g of sodium dihydrogen phosphate into a 1000-mL beaker. Dilute with water to 900 mL, and adjust with hydrochloric acid to a pH of 3.0 ± 0.1. Transfer the solution into a 1000-mL volumetric flask, and dilute with water to volume. Pass the solution through a 0.22-µm membrane filter.

Column storage solution
Mix 100 mL of methanol with 900 mL of water.

Test solution
Dilute Protein A to approximately 1 mg per mL with Mobile phase.

Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 280-nm detector and a 7.8-mm × 30-cm column that contains packing L33. Equilibrate the column for approximately 30 minutes at 0.5 mL of Mobile phase per minute or until a stable baseline is achieved.

Procedure
Separately inject 100 µL of each sample, and run the samples in the following sequence: Calibration standards, thyroglobulin, IgG, beta lactoglobulin, and lysozyme; the Standard solution; and the Test solution. Run the sequence three times isocratically using Mobile phase at 0.5 mL per minute for 30 minutes. Absorbance is detected at 280 nm. Analyze the 280-nm peak data, and pick the retention time (RT) with the largest peak area. Using the data from the Calibration standards, plot the mean RT versus the log molecular weight to produce the standard curve. The purity should be 95% in the main peak. Use the formula from the standard curve to give the log molecular weights of the Test solutions. Convert the log molecular weights of the Test solutions and the Standard solutions to actual molecular weights. The apparent molecular weight of protein A from the Standard solution is between 156 and 205 kDa; and the Protein A from the Test solution is within the same range.

Column cleaning and storage
Rinse the column with 100 mL of Column regeneration solution, and store by flushing with 100 mL of Column storage solution.

rPROTEIN A, C-CYS

C1478H2320N432O503S4

34317.5 Da

N-terminal Sequence AQHDEAQQNA

rProtein A, C-Cys is a recombinant Protein A lacking the C-terminal membrane binding part; instead, a C-terminal cysteine has been introduced for directed immobilization purposes. It has five homologous IgG binding domains identical to the native Protein A and is produced using Escherichia coli as the host cell followed by purification with conventional chromatography. rProtein A, C-Cys is manufactured as a bulk solution with an IgG-binding potency of greater than 95%. Because rProtein A, C-Cys is used as an ancillary material in the manufacture of recombinant therapeutic drugs, regulatory requirements differ from those for therapeutic drug products.

Packaging and storage
Store in closed containers at the temperature indicated on the label.

Labeling
Preserve in sealed containers, and store at a temperature of 20 or below.

Microbial limits 61
The total aerobic microbial count does not exceed 100 cfu per mL, and the total yeasts and molds count does not exceed 10 cfu per mL.

Bacterial endotoxins 85
It contains not more than 1 USP Endotoxin Unit per mg of total protein. [NoteThe Bacterial endotoxins test for rProtein A, C-Cys is used to describe the quality of this ancillary material. This test does not define the acceptable level of bacterial endotoxin in the preparation of injectable dosage forms in which rProtein A, C-Cys is used.]

in which A is the absorbance of rProtein A, C-Cys, at the wavelength of 275 nm and 0.22 is the molar absorptivity. Average the triplicate results, and determine a coefficient of variance (CV): the CV is 2.5%.

Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 214-nm detector and a 10-mm × 30-cm column that contains packing L54. Equilibrate the column with at least two column volumes of Mobile phase at a flow rate of 0.4 mL per minute.

Procedure
Inject 100 µL of Pretreatment solution, and allow the chromatography to continue for at least two column volumes. Repeat this twice before injecting 100 µL of the Test solution. Absorbance is detected at 214 nm. Integrate the main peak from the Test solution run and all other peaks not present in the Pretreatment solution runs. Calculate the percentage of impurities in the portion of the rProtein A, C-Cys taken by the formula:

100(ri / rs)

in which ri is the peak response for each impurity; and rs is the sum of the responses of all the peaks: the sum of all impurities is not more than 5%; and the Test solution shows a major peak at approximately 35 minutes.

rPROTEIN A

C1917H3039N565O658S3

44,618

N-terminal Sequence FLRPVE

Protein A is a component of the cell wall of Staphylococcus aureus. Recombinant Protein A (rProtein A) consists of five homologous immunoglobulin (IgG) binding domains (E, D, A, B, C) followed by a partial X domain sequence. It is expressed in Escherichia coli and purified via a column chromatography process. IgG columns are not used in the purification process. It is manufactured as a bulk solution with an IgG-binding potency greater than 95%. Release testing methods and specifications are described below. Because rProtein A is used as an ancillary material in the manufacture of recombinant therapeutic drugs, regulatory requirements differ from those of therapeutic drug products.

Packaging and storage
Store in closed containers at the temperature indicated on the label.

Labeling
The labeling states that the material is of recombinant DNA origin along with the lot number, storage conditions, and the statement Formulated in Water for Injection.

1X Running buffer
Prepare a solution of water and 20X Running buffer (19:1).

Gel staining solution
Prepare a solution of Coomassie brilliant blue R-250 (see Coomassie Brilliant Blue R-250 in Reagents under Reagents, Indicators, and Solutions) having a concentration of 0.5 g per L in a mixture of water, isopropanol, and acetic acid (6.5:2.5:1.0). Filter, and store at room temperature. Silver staining is not recommended.

Destaining solution
Mix 100 mL of acetic acid with 900 mL of water.

Standard preparation
Dilute USP rProtein A RS to 0.4 mg per mL with PBS solution. Further dilute this solution 1:1 with 2X Reducing sample buffer, and incubate in a closed tube for 5 minutes at 90. Mix, and quick spin prior to loading.

Test preparation
Dilute rProtein A with PBS solution to 0.4 mg per mL. Proceed as directed under Standard preparation beginning with Further dilute.

Comix solution
Dilute rProtein A and USP rProtein A RS with PBS solution to 0.8 mg per mL. This solution contains 0.4 mg per mL of each protein. Proceed as directed under Standard preparation beginning with Further dilute.

SDS-PAGE gel and apparatus set-up
Assemble gel apparatus following the manufacturers instructions. Lock the gel tension wedge in place, and fill approximately 200 mL of 1X Running buffer into the inside chamber. If there are no leaks, pour 600 mL of 1X Running buffer into the outer chamber. Gently pull the comb out of the cassette to immerse the wells in 1X Running buffer. Load 10 µL of each preparation as directed below under Gel loading onto a 10% Bis-Tris SDS-PAGE gel.4

Gel loading
Use the following gel loading scheme when running one Test preparation (see Table 1). Each Test preparation is run by itself and as part of the Comix solution that contains the rProtein A and USP rProtein A RS.

Table 1

Lane

Sample

Load Volume (µL)

LoadAmount (µg)

1

1X Sample buffer

10

N/A

2

MWM

20

N/A

3

Test preparation #1

10

2

4

Comix solution #1

10

4 (total)

5

Test preparation #1

10

2

6

1X Sample buffer

10

N/A

7

Standard preparation

10

2

8

MWM

20

N/A

9







10







Running the gel
Set the voltage to 125 volts, and run at a constant voltage. Run the gels until the bromophenol blue band is approximately 5 mm from the bottom of the gel (approximately 120 to 140 minutes).

Gel staining
Pour approximately 100 mL of Gel staining solution into the staining container. Place the gel into the staining container, and allow the stain to completely cover the gel. Cook the gel and container in a microwave for 30 seconds. Place the staining container on an orbital shaker, and stain the gel for 1 hour with gentle shaking.

Destaining
Drain the Gel staining solution, and add enough Destaining solution to the container to cover the gel. Place the container on an orbital shaker, and shake at low speed. Change the Destaining solution as necessary until a clear background is obtained. After destaining, rinse the gel thoroughly with water, and leave the gel in water for 10 minutes before scanning.

Gel scanning
Apply some water to the glass plate of the scanner, and place the gels on a wetted glass plate. Eliminate any bubbles. Using appropriate settings, scan the gels.

Data analysis
Choose a band between the 20 kD and 30 kD bands of the MWM to calculate the percentage of the retention factor. Draw a line in one lane (lane containing 1X Sample buffer) from the well to the apex (region of greatest intensity) of the chosen band.

The length of this line is denoted as the total distance (DT). For the lanes containing samples draw a line from the well to the apex of each band. For each band the length of this distance is the migration distance (DM) in mm. Record the DT and DM on the report sheet for each peak or band. The total distance should be the same for each lane on a gel. Calculate the percentage of the retention factor (RF) of each major peak or band, and document on the report sheet using the following equation:

%RF = DM / DT × 100

Also for each gel, record the number of bands and approximate molecular weight of each band in each sample.

System suitability
All bands between 20 kD and 70 kD are present. The lane containing 1X Sample buffer does not contain any bands.

Specificity
The rProtein A has one major band and a similar molecular weight that corresponds to those of the USP rProtein A RS. The Comix solution also shows a single major band.

B: IgG Binding[NoteThe IgG binding assay is a functional method for determining the percentage of rProtein A capable of binding to immobilized human polyclonal immunoglobulin. Since the percent of functional rProtein A in each lot is not less than 95%, the assay measures unbound protein versus total protein injected. This is done by comparing the absorbance in the flow-through to absorbance from an injection bypassing the column.]

Sample pretreatment (desalting)
In order to remove any buffer components that may contribute to absorbance in the unbound IgG column fraction, samples are desalted with Solution A. Desalting may be performed using a suitable desalting column5 depending on the volumes required.

IgG column
A 1-mL Sepharose column6 with immobilized human polyclonal IgG (hIgG) is required to perform this assay. [NoteThe IgG column requires washing when it is new, when it has performed several analysis cycles, or after system suitability failure. Column washing procedure is not required for each sample injection.]

Column washing solution A
Prepare a solution of 0.5 M acetic acid, pH 3.4 by adding 28.6 mL of acetic acid into a 1000-mL beaker, diluting to 900 mL with water, and adjusting with ammonium acetate to a pH of 3.4. Transfer the solution into a 1000-mL volumetric flask, and dilute with water to volume. Pass the solution through a 0.45-µm membrane filter.

Column washing solution B
Prepare a solution of 50 mM Tris, pH 7.6, 150 mM sodium chloride, and 0.05% Tween 20 by the following procedure. Add 6.06 ± 0.01 g of Tris and 8.77 ± 0.01 g of sodium chloride into a 1000-mL beaker. Dilute with water to 900 mL, and adjust with 0.5 M sodium hydroxide to a pH of 7.60 ± 0.05. Transfer the solution into a 1000-mL volumetric flask, and dilute with water to volume. Pass the solution through a 0.45-µm membrane filter (buffer solution). Add 0.5 mL of Tween 20 into 1 L of the buffer solution and mix thoroughly.

Solution A
Prepare a solution of 20 mM monobasic sodium phosphate and 150 mM sodium chloride, pH 7.6 by the following procedure. Add 2.76 ± 0.01 g monobasic sodium phosphate hydrate and 8.77 ± 0.01 g sodium chloride into a 1000-mL beaker. Dilute with water to 900 mL, and adjust with 5 M sodium hydroxide to a pH of 7.60 ± 0.05. Transfer the solution into a 1000-mL volumetric flask, and dilute with water to volume. Pass the solution through a 0.45-µm membrane filter.

Solution B
Prepare a solution of 100 mM phosphoric acid pH 2.8 by the following procedure. Add 6.8 mL of phosphoric acid into a 1000-mL beaker. Dilute with water to 900 mL, and adjust with 2 M potassium hydroxide to a pH of 2.80 ± 0.05. Transfer the solution into a 1000-mL volumetric flask, and dilute with water to volume.

Mobile phase
Use variable mixtures of Solution A and Solution B as directed for Chromatographic system. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation
Thaw USP rProtein A RS, and use directly.

Test preparation
Prepare a 4.0 to 6.0 mg per mL rProtein A solution in Solution A.

Chromatographic system
The liquid chromatograph is equipped with a 280-nm detector and a 1-mL column with immobilized hIgG. The chromatograph is equipped with a bypass valve to allow flow to be diverted from the column. Each analysis consists of a series of two injections, one where the sample is injected onto the column and one where the sample bypasses the column and flows directly into the detector. Perform three replicate analyses. The chromatograph is programmed as follows (see Table 2).

Table 2

Flow Rate (mL per minute)

Time (minutes)

Solution A (%)

Solution B (%)

Valve Position

Elution

1.0

06

100

0

column

re-equilibration

1.0

612

100®0

0®100

column

re-equilibration

1.0

1222

100

0

column

equilibration

0.4

2225

100

0

column

equilibration

0.4 (sample injected)

2535

100

0

column

isocratic

1.0

3549

100®0

0®100

column

regeneration

1.0

4963

0®100

100®0

column

re-equilibration

1

6365

100

0

bypass

equilibration

0.4

6568

100

0

bypass

equilibration

0.4 (sample injected)

6875

100

0

bypass

isocratic

Chromatograph the Standard preparation, record the peak responses, and calculate the percentage of hIgG binding as directed for Procedure: the percentage of hIgG binding 95% and the relative standard deviation for replicate analysis is not more than 1%.

Procedure
Inject a volume (about 100 µL) of the Test preparation. Record the chromatogram, and measure the peak responses. Calculate the percentage of hIgG binding activity by the following formula:

100  100(rC / rB)

in which rC is the unbound material peak response from the column injection and rB is the bypass peak response from the bypass injection. Each replicate analysis of the Test preparation is not less than 95% of hIgG binding. Report the average value from three replicate analyses.

Microbial limits 61
The total aerobic microbial count does not exceed 100 cfu per mL, and the total yeasts and molds count does not exceed 10 cfu per mL.

Bacterial endotoxins 85
It contains not more than 0.5 USP Endotoxin Unit per mg of total protein. [NoteThe Bacterial endotoxins test for rProtein A is used to describe the quality of this ancillary material. This test does not define the acceptable level of bacterial endotoxin in the preparation of injectable dosage forms in which rProtein A is used.]

in which A is the absorbance of rProtein A at the wavelength of 275 nm and 0.165 is the molar absorptivity. Average the triplicate results, and determine a coefficient of variance (CV): the CV is 5%.

UV spectral analysis
Dilute rProtein A to 1 mg per mL in Water for Injection. Using a scanning UV spectrophotometer and Water for Injection as the blank, obtain spectral scans over the range of 240 to 360 nm. From the resulting data, calculate the absorbance value at 270 nm and the ratio of absorbance at 270 to 250 nm (i.e., E270/E250): the absorbance at 270 nm of a 1 mg per mL solution of rProtein A in Water for Injection is within the range 0.140.20.

Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 214-nm and 280-nm detector and a 9.4-mm × 25-cm column that contains packing L35. The flow rate is 1 mL per minute. Chromatograph the Standard solution as directed for Procedure: rProtein A shows a single major peak at approximately 9 minutes and the area percentage is 98% at 214 nm and 95% at 280 nm.

Procedure
Inject 100 µL of the Test solution into the chromatograph, run isocratically for 15 minutes, and record the chromatogram. The values for the rProtein A from the Test solution correspond to the specifications of the USP rProtein A RS from the Standard solution.

pI Markers
Use a suitable marker set containing markers between 3 and 10.7

IEF gel
Use a suitable gel with the range of between 3 and 10 and a size of 100 × 125 mm.8

Procedure
Apply 5-µL aliquots of the pI Markers, Test solution, and the Standard solution to the IEF gel, and run under 1W of power for approximately 10 minutes. Remove the sample mask, and apply power with concurrent cooling between 5 to 10 of the focusing chamber for 40 minutes at a setting of 1000V, 20 mA, 25W. Fix the IEF gel for 1 hour in 20% trichloroacetic acid, then stain using a suitable stain for IEF gels.9 Finally, wash and dry the gel: the correlation coefficient of the best fit line for the pI Markers versus their migration in cm is 0.990, and the rProtein A from the Standard solution shows a single major band within the pI range of 4.6 to 5.2. A single band is seen in the Test solution that corresponds to the pI range of the Standard solution.

Limit of Triton X-100

Mobile phase
Prepare a filtered and degassed mixture of water and acetonitrile (60:40).

Triton X-100 spike solution
Combine 5 mg per mL of USP rProtein A RS and 0.15% Triton X-100 (9:1) to obtain a solution having known concentrations of rProtein A and 0.015% Triton X-100.

Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 214-nm and a 280-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L11. The flow rate is 1 mL per minute. Chromatograph the Triton X-100 spike solution as directed for Procedure: Triton X-100 has a single major peak at approximately 9 minutes, and the rProtein A shows a smaller or undetectable peak at the same retention time.

Procedure
Inject about 100 µL of the Test solution into the chromatograph, run isocratically for 35 minutes, and record the chromatogram. The absorbance is detected at 223 nm: the Triton X-100 peak is not more than 0.015% (equivalent to the Triton X-100 spike solution).

rPROTEIN A, B4, C-CYS

C1177H1854N326O384S1

26747.6 Da

N-terminal Sequence AQGTVDAKFD

rProtein A, B4, C-Cys is a recombinant protein derived from the B-domain of Protein A. The Protein A domain has been alkali-stabilized by site-specific mutagenesis and multimerized to a tetramer with a C-terminal cysteine for directed immobilization purposes. rProtein A, B4, C-Cys is produced using Escherichia coli as the host cell followed by purification with conventional chromatography. rProtein A, B4, C-Cys is manufactured as a bulk solution with an IgG-binding potency of greater than 95%. Because rProtein A, B4, C-Cys is used as an ancillary material in the manufacture of recombinant therapeutic drugs, regulatory requirements differ from those for therapeutic drug products.

Packaging and storage
Store in closed containers at the temperature indicated on the label.

Labeling
Preserve in sealed containers, and store at a temperature of 20 or below.

Microbial limits 61
The total aerobic microbial count does not exceed 100 cfu per mL, and the total yeasts and molds count does not exceed 10 cfu per mL.

Bacterial endotoxins 85
It contains not more than 1 USP Endotoxin Unit per mg of total protein. [NoteThe Bacterial endotoxins test for rProtein A, B4, C-Cys is used to describe the quality of this ancillary material. This test does not define the acceptable level of bacterial endotoxin in the preparation of injectable dosage forms in which rProtein A, B4, C-Cys is used.]

Procedure
Prepare triplicate samples for analysis. Measure the absorbance of each Test preparation at 275 nm after correcting for the absorbance using the Formulation buffer solution as the blank. Determine the protein concentration using the equation:

Protein concentration (mg per mL) = (A275 / 0.22)

in which A is the absorbance of rProtein A, B4, C-Cys, at the wavelength of 275 nm and 0.22 is the molar absorptivity. Average the triplicate results, and determine a coefficient of variance (CV): the CV 2.5%.

Chromatographic system (see Chromatography 621)
The liquid chromatograph is equipped with a 214-nm detector and a 10-mm × 30-cm column that contains packing L54. Equilibrate the column with at least two column volumes of Mobile phase at a flow rate of 0.4 mL per minute.

Procedure
Inject 100 µL of Pretreatment solution, and allow the chromatography to continue for at least two column volumes. Repeat this twice before injecting 100 µL of the Test solution. Absorbance is detected at 214 nm. Integrate the main peak from the Test solution run and all other peaks not present in the Pretreatment solution runs. Calculate the percentage of impurities in the portion of rProtein A, B4, C-Cys taken by the formula:

100(ri / rs)

in which ri is the peak response for each impurity; and rs is the sum of all the responses of all the peaks: the sum of all impurities is not more than 5%; and the Test solution shows a major peak at approximately 37 minutes.

1S (USP31)

1
A suitable enzyme immunoassay kit is available from TECRA International Pty Ltd., Australia (No. SETVIA96).